NO330512B1 - Use of 4-H-1-benzopyran-4-one derivatives for the preparation of a pharmaceutical preparation for the inhibition of smooth muscle cell proliferation - Google Patents

Description

The field of the invention

The present invention relates to the use of 4-H-1-benzopyran-4-one derivatives for the production of a pharmaceutical preparation for inhibiting smooth muscle cell (SMC) proliferation.

The background of the invention

The cellular response to vascular injury - cellular dysfunction, activation, dedifferentiation, proliferation and migration - culminates in clinical events such as restenoses, which occur after balloon angioplasty and placement of stents for the treatment of human atherosclerotic disease \ Smooth muscle cell (SMC) proliferation is a common and possibly uniform feature of models of vascular injury, and SMCs are the major cellular component of neointimal lesions<2>'<3>. New interest in inhibiting SMC proliferation has followed the increase in the use of stents in the treatment of coronary disease, since stent restenosis is almost exclusively dependent on neointimal formation and SMC hyperplasia<4>. It is estimated that as many as 100,000 patients with stent restenosis required treatment in 1997 alone<5>; therefore, an easily administered effective inhibitor of SMC hyperplasia would have major clinical and economic implications<6>.

Attempts to inhibit SMC proliferation in models of vascular injury, either by modulating cell mediators of the proliferative response, or by directly interfering with the cell cycle machinery, have led to important insights into neointimal formation. Cell cycle progression is a tightly controlled event regulated positively by cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits<7>, and negatively by Cdk inhibitors and tumor suppressor genes such as retinoblastoma protein ((Rb) and p53<8>. Adenovirus-mediated overexpression of endogenous Cdks -inhibitors p21 and ^ 27^ or of a constitutively active form of Rb block neointimal formation in a rat carotid injury model9-1 1; similarly, inhibition of the active transcription factor E2F by competitive overexpression of similar DNA binding sites also inhibits SMC proliferation and neointimal formation <12> Such studies support the general hypothesis that cell cycle inhibition is an attractive target for intervention in vascular lesion formation.

While interventions have aided the analysis of the mechanisms that regulate neointimal formation, they suffer from the inadequacy of currently not being clinically suitable for the treatment of human vascular disease. A water-soluble, low molecular weight compound with special cell cycle regulatory effects, particularly one with oral activity, would have wide application both experimentally and possibly clinically. The recently identified flavone, flavopiridol, is a Cdk inhibitor that strongly blocks the activity of Cdk2, Cdc2, and CdLt<13>"<16>.1 unlike other pharmacological inhibitors of Cdk, flavopiridol excels in its kinase specificity, its oral availability, and its potency by being effective at nanomolar concentrations<16>.These unique features result in an advantageous side effect profile that has led to the trial of flavopiridol in phase 1 clinical trials for the treatment of refractory neoplasms 17. Given these properties, the ability of flavopiridol to inhibit SMC proliferation in vitro and after balloon injury of the rat carotid artery now investigated. It is found that flavopiridol is a potent and selective inhibitor of cell cycle progression and that it arrests SMC proliferation both in vivo and in vitro; moreover, neointimal formation is effectively blocked by oral doses of flavopiridol lower than those known to have toxic effects in humans.

It has now surprisingly been found that 4-H-1-benzopyran-4-one derivatives are suitable SMC proliferation inhibitors. It is known that 4-H-1-benzopyran-4-one derivatives are suitable for controlling tumors. However, it is surprising that 4-H-1-benzopyran-4-one derivatives used in the present invention effectively act as an SMC proliferation inhibitor at dose levels lower than the dose levels used in controlling tumor growth.

Consequently, an aim according to the invention is to use 4-H-1-benzopyran-4-one derivatives as inhibitors of smooth muscle proliferation.

Figure 1. Effect of flavopiridol on HASMC DNA synthesis. A. Resting HASMC were treated in the absence (−) or presence (+) of bFGF (10 ng/ml) and with the indicated concentrations of flavopiridol (nmol/1) for 24 h. BrdU incorporation as a measure of proliferation was determined by an ELISA-based assay and expressed as percent incorporation in the absence of bFGF treatment.<*>p < 0.05 relative to untreated cells.

■ fp < 0.05 compared to treatment with bFGF in the absence of flavopiridol. B. HASMC were treated with bFGF (10 ng/ml), thrombin (2U/ml), or vehicle in the presence or absence of flavopiridol (75 nmol/l) and BrdU incorporation was measured. * p < 0.05 compared to untreated cells. ** p< 0.05 in relation to treatment with bFGF alone, f< 0.05 in relation to treatment with thrombin alone.

Figure 2. Effect of flavopiridol on HASMC proliferation. Resting HASMC were treated with bFGF (10 ng/ml) alone (□), bFGF and flavopiridol (75 nmol/l) (□), or vehicle (□) for the indicated time and cell numbers after treatment were determined. Results are expressed as cell count per well (x IO<3>).

Figure 3. Effect of flavopiridol on cyclin-dependent kinase activity in HASMC. Resting HASMC were treated with bFGF (10 ng/ml), thrombin (2 U/ml) or vehicle in the presence or absence of flavopiridol (75 nmol/l), and phosphorylation of histone H1 was quantified as a measure of Cdk activity and expressed as a percentage of Cdk activity in the absence of bFGF treatment. * p < 0.05 compared to untreated cells. ** p < 0.05 in relation to treatment with bFGF alone, f < 0.05 in relation to treatment with thrombin alone. Figure 4. Regulation of cell cycle-related proteins by flavopiridol. Resting HASMC were treated in the presence (+) or absence (-) of bFGF (10 ng/ml), thrombin (2 U/ml) and/or flavopiridol (75 nmol/l) for 24 hours. Immunoblotting of cell lysates was performed with specific antibodies that recognize cyclin Di (upper part), PCNA (middle part) and phosphorylated (pRb) and hyperphosphorylated (ppRb) Rb (lower part). Figure 5. Effects of flavopiridol on MAP kinase activity in HASMC. Resting HASMC were treated in the presence (+) or absence (-) of bFGF (10 ng/ml), thrombin (2 U/ml), PD98059 (30 nmol/1) and/or flavopiridol (75 nmol/1) for 30 minutes. Levels of phosphorylated Erk1 (pErk1) and Erk2 (pErk2) were measured by immunoblotting with a phosphorylation-specific antibody that recognizes both proteins (upper part). MAP-kinase activity was measured with an "in-gel" kinase assay, which uses myelin basic protein as a substrate (lower part). Figure 6. The viability of HASMC after treatment with flavopiridol. Resting HASMC were treated with flavopiridol (75 nmol/l), TNF-α (50 ng/ml), or vehicle for the time periods indicated. Cell viability was assessed by trypan blue exclusion. The results are expressed as a percentage of viable cells in relation to the total cell count. Figure 7. Inhibition of neointimal formation in rat carotid artery by flavopiridol after balloon injury. The neointima/media ratios were measured in histological sections of rat carotid arteries treated with or without flavopiridol (5 mg/kg) for 5 days after injury. The arteries were examined 7 (n = 12) and 14 (n = 12) days after injury. The percentage of PCNA-positive nuclei (± SEM, expressed as a percentage of the number of nuclei counted) in the neointima of the arteries from each time point and treatment type is also given. * p < 0.05 compared to treatment with vehicle. Figure 8. Histological sections from rat carotid arteries. The sections are from the arteries 7 (A and B) and 14 (C and D) days after injury. The arteries shown in A and C are from rats treated with flavopiridol (5 mg/kg) by gavage; the arteries in B and D are from rats treated with vehicle alone. Original magnification x 100. Figure 9. Cdk2 expression after balloon injury in rat carotid arteries. The sections are four arteries 7 (A and B) and 14 (C and D) days after injury. The arteries shown in A and C are four rats treated with flavopiridol (5 mg/kg) by gavage; the arteries in B and D are from rats treated with vehicle alone. Cdk2-positive nuclei, located mainly in the neointima, are stained with vector blue according to the alkaline phosphatase method. Original magnification x 100.

The present invention therefore encompasses the use of a compound of formula Ia

R5

IN

OH O Formula Ia

where R 1 is hydrogen, C 1 -C 3 alkyl, naphthyl, phenyl; phenyl mono- or polysubstituted with halogen, Ci-C4-alkyl, Ci-C4-alkoxy, hydroxyl, carboxyl, COO-Ci-C6- alkyl, CONH2, CONH-C1-C6- alkyl, CON(Ci-C6-alkyl) 2, nitro, trifluoromethyl, amino, C 1 -C 4 -alkylamino, di-C 1 -C 4 -alkylamino, or phenyl; pyridyl or thienyl;

R 2 is hydrogen or C 1 -C 3 alkyl;

R5 is C1-C3-alkyl, C3-C5-cycloalkyl, or C3-C5-cycloalkyl-C1-C4-alkyl,

or a pharmaceutically acceptable acid addition salt thereof, for the preparation of a pharmaceutical preparation for inhibiting smooth muscle proliferation, wherein the dose of the compound according to formula Ia is less than 70%, preferably less than 60%, in

in particular less than 50% of the dosage required to control tumor growth.

The compounds used according to the invention have two asymmetric centers, one where the heterocyclic ring containing nitrogen is fused to the benzopyran unit (C-4'), the

others at the carbon atom bearing the OH group on the same ring (C-3'), which means that two pairs of optical isomers are possible. The definition of the compound according to the invention includes all possible stereoisomers and their mixtures. In particular, it includes racemic forms and the isolated optical isomers with special activity. The two racemates can be dissolved by physical methods such as, for example, fractional crystallization. The individual optical isomers can be obtained from the racemates by conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization. Examples of alkyl groups suitable for R 1 to R 5 are straight-chain or branched radicals.

Suitable examples of salts of the compounds used according to the invention with inorganic or organic acids are hydrochloride, hydrobromide, sulphate, phosphate, acetate, oxalate, tartrate, citrate, maleate or fumarate.

Particularly preferred is the use of compounds of formula Ia, where Ri is phenyl, thienyl, pyridyl, chlorophenyl, dichlorophenyl, methylphenyl, aminophenyl, bromophenyl, hydroxyphenyl or naphthyl;

R 2 is hydrogen and

R 5 is methyl.

A particularly important compound is (-)-cis,-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxyl-methyl)-pipeirdinyl]-4H-benzopyran-4-one ( Flavopiridol), especially in the form of hydrochloride.

The compounds used according to the invention can be prepared according to the description in U.S. Pat. Patent No. 4,900,727 and U.S. Pat. Patent No. 5,284,856. The examples in these U.S. Pat. patents are also important for the present invention.

The compounds used according to the present invention inhibit smooth muscle cell proliferation. They may therefore find use as pharmaceutical preparations for inhibition of smooth muscle cell proliferation, containing at least one compound of formula Ia, defined above or at least one of its pharmaceutically acceptable acid addition salts. Typical areas of application for the compounds used according to the invention are diseases/disorders/injuries associated with vascular lesions that are rich in smooth muscle cells. A very important example is therefore lesions after balloon injuries. Another important area of application is the prevention of restenosis after implantation of stents.

For the pharmaceutical preparations obtained by the invention, an effective amount of the aforementioned active substance is used, either per see or possibly in combination with suitable pharmaceutical excipients in the form of tablets, coated tablets, capsules, suppositories, emulsions, suspensions or solutions, where the content of active compound is up to 95%, preferably between 10 and 75%.

The professional will know which excipients are suitable for the desired formulation of the pharmaceutical preparation because this is within his area of knowledge. In addition to auxiliaries for tablets or solvents, gel forms, bases for suppositories and other excipients for the active substance, it is possible to use, for example, antioxidants, dispersants, emulsifiers, anti-foaming agents, flavourings, preservatives, solubilizing agents or colourings.

The active substance can be administered orally, parenterally, intravenously or rectally, oral administration being advantageous. For a form of oral administration, the active substance can be mixed with other compounds together with the excipients suitable in this regard, such as excipients, stabilizers and inert diluents, and conventional methods can be used to transfer it to suitable administration forms, such as tablets, coated tablets, hard gelatin capsules and aqueous alcoholic or oily suspensions or solutions. Examples of inert excipients that can be used are gum arabic, magnesium oxide, lactose, glucose or starch, in particular corn starch. In this context, the formulation can be prepared as dry granules or moist granules. Examples of suitable oil excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil.

With subcutaneous or intravenous administration, a solution, suspension or emulsion of the active substance is formed, if necessary, substances that are conventional for this purpose are used, such as solubilizing substances, emulsifiers and other auxiliary substances. Examples of suitable diluents are water, physiological sodium chloride solution or alcohols, for example ethanol, propanol or glycerol, and also sugar solutions such as glucose solutions or mannitol solutions, or a mixture of the various solvents mentioned.

The dose of 4H-1-benzopyran-4-one derivatives that can be administered daily is selected to be adapted to the desired effect. The 4H-1-benzopyran-4-one derivatives can be administered in a dose that is less than 70%, preferably less than 60%, in particular less than 50% of the dose used to control tumor growth in the individual mammal. An example would be - in a nude mouse - xenograft model - a dose of approx. 5 mg/kg body weight administered orally once daily. This is half the dose that inhibits tumor growth in the same animal model (Drees et al. Clin. Cancer Res. 1997; 3; 273-279).

The pharmacokinetic properties of the 4H-1-benzopyran-4-one derivatives may make it necessary to administer the compound several times a day or to choose slow release formulations.

Examples

1. Flavopiridol inhibits smooth muscle cell proliferation and neointimal formation in vivo in a rat carotid model of vascular injury.

The well-established rat carotid injury model, in which neointimal lesion formation following catheter-induced injury is critically dependent on SMC proliferation (Clowes et al. Lab. Invest. 1983; 49:327-333, Clowes et al. Circ. Res. 1985; 56: 139-145) was used to investigate whether flavopiridol induces growth arrest in SMC in vivo, as it does in vitro. Flavopiridol was administered orally at a dose of 5 mg/kg once daily, beginning on the day of injury and continuing for 4 days thereafter, since this time period covers the initial induction of Cdk2 and the first wave of SMC proliferation in this model (Circ. Res. 1995; 77:445-465, Circ. Res. 1997; 80:418-426). Mean intimal and medial areas were quantified 7 and 14 days after injury, and neointimal lesion size was expressed as the ratio of neointimal to medial area. The treated and untreated group consisted of 12 animals. The ratio at day 7 was 1.00 ± 0.05 in the arteries of vehicle-treated rats, and 0.65 ± 0.04 in flavopiridol-treated rat arteries, a reduction of 35%. On day 14, the neointimal/medial ratio was 1.08 ± 0.04 in vehicle-treated rats, and 0.66 ± 0.03 in flavopiridol-treated rats, a reduction of 38.9%. These effects were statistically significant at both time points (P < 0.05).

Methods

Materials - Flavopiridol (L86-8275, (-)- cis, -5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl]-4H-benzopyran -4-one) was obtained from Hoechst Marion Roussel, Inc., and was dissolved in dimethyl sulfoxide as a 50 mmol/l stock solution for cell culture experiments or in water for in vivo experiments. Basic fibroblast growth factor (bFGF) was supplied from Collaborative Biochemical and thrombin from Sigma. The MEK1 inhibitor PD98059 was obtained from New England Biolabs.

Cell culture - Human aortic smooth muscle cells (HASMC) were obtained from Clonetics and were cultured as previously described<18>. Cells were used at passages 5-9. Before experiments were performed, growth was arrested at 80% confluence for 48 h with medium containing 0.2% fetal bovine serum.

Cell proliferation ELISA - Cell proliferation was measured by ELISA (Amersham Life Science). HASMC were cultured in gelatin-coated 96-well plates and made quiescent. Cells were treated with 10 ng/ml bFGF, 2 U/ml thrombin, or vehicle for 24 h. Flavopiridol (75 nmol/l) was administered 1 hour before growth factor treatment. 5-bromo-2'-deoxyuridine (BrdU) was added to a final concentration of 10 pmol/l during the last 2 hours of the experiment. BrdU incorporation was measured as described<19>. The results are expressed as mean ± standard error for 12 samples per condition for two independent experiments.

Cell Count - Arrested HASMCs, grown to 50% confluence in 6-well plates were treated with or without flavopiridol (75 nmol/1) or bFGF (10 ng/ml). At intervals after treatment, cells were trypsinized and cell counts determined using a hemocytometer.

Western blot analyzes - Resting HASMC were treated in the presence or absence of growth factors and/or flavopiridol as indicated. Western blot analyzes were performed as previously described<18>. The primary antibodies were: A polyclonal antihuman cyclin D1 antibody (M-20, Santa Cruz), a monoclonal antihuman proliferating cell nuclear antigen (PCNA) antibody (PC 10, Sigma), a phosphorylation-specific p44/42 (Erk1/Erk2) MAP -kinase monoclonal antibody (New England Biolabs), and an anti-Rb monoclonal antibody (G3-245, Pharmigen), which recognizes the phosphorylated (pRb) and the highly phosphorylated (ppRb) Rb types. For immunoblotting studies, the experiment was repeated at least three times.

Cdk activity - Resting HASMC were treated with agonists and inhibitors for 24 h and total cell lysate was prepared as described for Western blotting. The kinase assay was performed with a histone and kinase assay kit (Upstate Biotechnology) according to the manufacturer's instructions. Briefly, 10 µl of peptide inhibitors for protein kinase C (2 pmol/1) and protein kinase A (2 pmol/1), 100 µg of cell lysate, 10 µl of assay buffer and 10 µl of a mixture containing 75 pmol/1 magnesium chloride, 500 pmol/ 1 ATP and 1 pCi/ml [γ -<32>P]ATP mixed in a microcentrifuge tube. After incubation at 30°C for 10 minutes, aliquots were pipetted onto phosphor cellulose paper. The papers were washed in 0.75% phosphoric acid, followed by measurements of cpm in a scintillation counter (Beckman). Results are expressed as mean ± standard error of 3 samples and are representative of three independent experiments.

"In-Gel" Kinase Assay - Resting HASMC were treated with growth factors for 30 minutes and total cell lysate was prepared as described for Western blotting. In some experiments, HASMC were pretreated for 60 min with 30 nmol/l PD98059, flavopiridol, or vehicle. Equal amounts of proteins (50 ng/field) were resolved on a polyacrylamide gel that was copolymerized with 350 ng/ml basic myelin protein. The gel was treated with [γ-32P]ATP and autoradiography was performed as described<19>.

Trypan blue exclusion - HASMC were grown in 5-cm dishes at low confluence and growth arrested as described. Cells were treated with flavopiridol (75 nmol/l) or tumor necrosis factor-α (TNF-α; 50 ng/ml) for the indicated time points. After removal of the medium, 0.4% trypan blue in phosphate-buffered saline was added to the dishes. After 5 minutes, the cells in the dish were counted under a microscope. Blue cells were counted as non-viable cells.

Rat carotid injury model - Injury to the rat carotid artery was performed essentially as described. Adult male Sprague-Dawley rats (400-500 g, Zivic-Miller) were anesthetized with an intraperitoneal injection of ketamine (2 mg/kg) and xylazine (4 mg/kg). The left internal carotid was then cannulated with a 2F embolectomy catheter. The balloon was inflated with saline and pulled through the artery three times to produce an expanding and exposing lesion. The right carotid artery remained uninjured and served as an injury control for each animal. Immediately after reconstitution from anesthesia and for 4 consecutive days, the rats were administered flavopiridol (5 mg/kg in water) or water by gavage as a blind test. All the rats survived the surgery and there were no obvious signs of toxicity associated with the drug administration at the doses used. At separate times after the carotid injury, the rats were anesthetized as above and perfusion-fixed systemically with 4% paraformaldehyde in phosphate-buffered saline. Right and left carotid arteries were removed and distended by injection of 4% paraformaldehyde through the lumen, after which they were dehydrated and stored in 70% ethanol at 4 °C. Immunohistochemistry was performed as described previously<18>, using the monoclonal PCNA antibody and a polyclonal antihuman Cdk2 antibody (M2-G, Santa Cruz).

Image Analysis - The outermost distal and proximal regions of each artery (approximately 500 pm) were removed. Ten intermediate cross-sections (8 µm each) taken at 500 µm intervals were analyzed for each artery. Sections were fixed and stained with hematoxylin and eosin as previously described<18>. Using a Nikon Diaphot 300 microscope and a 4x objective, each cross-section was captured as a digital image using a Hamamatsu C5985 video camera and TCPro 2.41 (Coreco, Inc.). Medial and neointimal areas were determined using NIH Image software. Medial and neointimal borders were determined by one section reader (A.M.) and confirmed in a blinded manner by another reader (C.P.). Lesion size was expressed as the neointima/media ratio. Results for each group were expressed as mean ± standard error. 92% or more of the images could be deciphered in each group; the rest were not analyzed because fixation artifacts.

Statistical analyzes - If appropriate, data from quantitative studies were expressed as mean ± standard error. For multiple treatment groups, a factorial ANOVA followed by Fisher's least significant difference test was used. Statistical significance was accepted at p < 0.05.

Results

Flavopiridol inhibits HASMC proliferation - On the basis of flavopiridol's ability to inhibit proliferation of various tumor cell lines, the hypothesis that its use would block the growth of primary culture of human SMCs was investigated. Growth-arrested HASMCs were treated with the SMC mitogen bFGF (10 ng/ml) for 24 h in the presence of increasing concentrations of flavopiridol and proliferation was measured by an ELISA-based assay. Compared to untreated cells, the proliferation of bFGF-treated cells increased 5.4-fold (Figure 1A). Pretreatment for 1 hour with as little as 50 nmol/1 flavopiridol significantly reduced HASMC proliferation (to 3.9-fold, /> < 0.05), an effect that was approximately maximal at concentrations of 75 nmol/1. Similar results were obtained using thymidine uptake as an independent measure of DNA synthesis (not shown).

To examine the generality of flavopiridol's effect on SMC proliferation, its effect on mitogenesis elicited by thrombin (2 U7ml), which acts through a G protein-coupled receptor, similar to bFGF, which stimulates a member of the receptor tyrosine kinase family, was examined. Flavopiridol (75 nmol/1) significantly and potently inhibited both bFGF- and thrombin-induced HASMC proliferation (5.4-fold vs. 1.8-fold and 2.4-fold vs. 0.7-fold, respectively, p < 0.05, Figure 1B). Cell counts were performed to confirm that the effect of flavopiridol on cell cycle progression in HASMCs indeed reflected changes in proliferation. bFGF (10 ng/ml) induced a 3-fold increase in cell number after 3 days of treatment (Figure 2). Corresponding results were shown in the ELISA-based assays, flavopiridol (75 nmol/1) effectively blocked bFGF-induced proliferation.

Flavopiridol inhibits Cdk activity and cell cycle-related gene expression in HASMC - To assess the specific effect of flavopiridol on the cell cycle machinery, the histone H1 kinase activity in cell lysates from growth factor-stimulated HASMC was measured. Phosphorylation of histone Hl reflects the activities of Cdc2 and Cdk2<20>. Treatment of HASMC with bFGF and thrombin resulted in 4.4-fold and 3.6-fold increases in histone H1 kinase activity, respectively (Figure 3). These increases in cyclin-dependent kinase activity were completely blocked by pretreatment with flavopiridol (75 nmol/1).

Western blot analysis also investigated whether flavopiridol was affected

growth factor-induced regulation of cell cycle-related proteins in HASMC. Cyclin Di is a Gi-phase cyclin that is upregulated upon growth factor stimulation and rapidly degraded upon removal from the cell cycle 21. Cyclin Di protein levels were upregulated, 6.3-fold and 3.2-fold, respectively, in response to bFGF and thrombin treatment for 24 hours (Figure 4), an effect that could be completely blocked by pretreatment with flavopiridol. Similarly, increase in expression of PCNA, which is mainly synthesized during S-phase in connection with DNA replication<22>, was also blocked by flavopiridol pretreatment. As a final measure of cell cycle-related proteins, Rb phosphorylation in response to growth factor expression using an antibody that recognizes phosphorylated Rb was examined. Rb is a cell cycle regulator that binds to, and inactivates, the transcription factor E2F when Rb is in an unphosphorylated state<23>and induces SMC growth arrest in vivo n. Phosphorylation inactivates Rb and allows progression through S phase to continue. Analysis of Rb phosphorylation is particularly relevant because Rb is a target for Cdk2 and Cdktin in vivo. Both thrombin- and bFGF-induced hyperphosphorylation of Rb was an effect that was inhibited by flavopiridol. Overall, these results suggest that flavopiridol

affects expression and activity of Gi and S-phase-related cell cycle control elements in HASMC associated with its growth inhibitory effects.

Flavopiridol has no effect on MAP kinase phosphorylation or activity - To ensure that flavopiridol acts specifically at the cell cycle level, rather than nonspecifically on upstream kinase pathways, phosphorylation and activity of Erk1 (p44 MAP kinase) and Erk2 (p42 MAP kinase ), two members of the MAP kinase family, measured. These kinases were chosen because they are located immediately upstream of the transcriptional events that occur in response to growth stimuli and downstream of a number of critical mitogenic signaling pathways<24>. An intact response of MAP kinases suggests that the upstream mitogenic pathways are also intact. The phosphorylation status of Erk1 and Erk2 was measured with a monoclonal antibody that specifically recognizes the phosphorylation - and - consequently activated forms. As a control for these experiments, PD98059, a potent and selective inhibitor of MAP kinase activity, was used 25. Increased amounts of phosphorylated Erk1 and Erk2 were detected after treatment of HASMC with thrombin and bFGF

for 30 min, compared to untreated cells (Figure 5, upper part). Phosphorylation of Erk1 and Erk2 by both thrombin and bFGF was blocked by pretreatment with PD98059, but not with flavopiridol. To confirm these findings, Erk1 and Erk2 activity was measured by an "in-gel" kinase assay (Figure 5, lower part). Again, Erk1 and Erk2 activities were found to increase in response to thrombin and bFGF, an effect that could be inhibited by PD98059 but not by flavopiridol. These experiments along with those represented in Figures 3 and 4 provide evidence that the effects of flavopiridol on HASMC proliferation are caused by a specific arrest of the cell cycle machinery by blocking Cdk activity without affecting upstream signaling events.

Flavopiridol does not increase HASMC viability - Previous reports regarding flavopiridol activity in other cell types have shown that, depending on the cell type, flavopiridol can either induce growth arrest without affecting viability, or it can cause apoptosis<16>'<26>'<29>. It was therefore assessed whether flavopiridol reduced the viability of HASMC by measuring trypan blue exclusion at different time points after treatment. Resting HASMC were treated with flavopiridol (75 nmol/1), vehicle or TNF-α (50 ng/ml), a cytokine known to induce apoptosis in this cell type<30>. While TNF-α strongly reduces the viability of HASMC, resulting in the death of essentially all cells treated for 24 h, flavopiridol had no such effect (Figure 6). It was observed that with higher concentrations and longer incubations some reduction in viability in the presence of flavopiridol could occur

(not shown). However, under the conditions investigated, flavopiridol mainly induces growth arrest, without affecting SMC viability.

Flavopiridol inhibits smooth muscle cell proliferation and neointimal formation in vivo in a rat carotid injury model of vascular injury - The well-established rat carotid injury model was used in which formation of neointimal lesions after catheter-induced injury is critically dependent on SMC proliferation<2>'<3>, to investigate whether flavopiridol induces growth arrest of SMC in vivo, as it does in vitro. Flavopiridol was administered orally at a dose of 5 mg/kg once daily, beginning on the day of injury and continuing for the next 4 days, since this time period covers the initial injection of Cdk2 and the first wave of SMC proliferation in this model<31>'< 32>. Average intimal and medial areas were quantified 7 and 14 days after injury, and neointimal lesion size was expressed as the ratio of the neointimal area to the medial area. There were 12 animals in both the treated and untreated groups. The neointimal/medial ratio at 7 days was 1.00 ± 0.05 in the arteries of vehicle-treated rats, and 0.65 ± 0.04 in flavopiridol-treated rat arteries, a reduction of 35.0% (Figure 7). On day 14, the neointimal/medial ratio was 1.08 ± 0.04 in vehicle-treated rats, and 0.66 ± 0.03 in flavopiridol-treated rats, a reduction of 38.9%. These effects were statistically significant at both time points ( p < 0.05). Representative artery sections are shown in Figure 8.

To directly demonstrate that flavopiridol inhibits smooth muscle cell proliferation, sections were stained for PCNA expression in representative areas of each artery and the percentage of PCNA-positive nuclei in the neointima was determined. On day 7, 31.1 7.2% of cores in injured areas from untreated rats were PCNA-positive, whereas only 11.8 ± 1.5% of injured arteries in flavopiridol-treated rats were PCNA-positive (Figure 1; p < 0 .05). At day 14, PCNA-positive nuclei were present in 10.4 ± 2.0% of neointimal cells from treated but only in 4.2 ± 0.5% of neointimal cells from untreated, uninjured rat arteries ( p < 0.05 ). (PCNA-positive nuclei were rarely seen in uninjured arteries, regardless of treatment.) Similarly, Cdk2-positive cells were far less common in the neointima of flavopiridol-treated rats (Figure 9, A and Q, compared with arteries from untreated rats (B and D) , at both 7 and 14 days after injury.

Discussion

In the present study, it has been investigated whether the new Cdk inhibitor, flavopiridol, the most potent and specific inhibitor of Cdk known, is a suitable candidate for inhibiting SMC proliferation in vivo, especially in the area of vascular damage. Previous attempts to control the cell cycle machinery therapeutically in the treatment of vascular diseases have produced a rationale for the present studies<9>"<12>; however, methods used until now are based on gene transfer technology to inhibit cell cycle progression. Major clinical difficulties prevent for time application of these techniques<33>. During these studies, it was reported that CVT-313, a newly identified compound that also has Cdk-inhibitory properties, but at micromolar concentrations, can also inhibit neointimal formation; however, it was necessary that CVT- 313 was infused into the carotid artery at the time of injury to produce this effect<34>. In contrast, it was shown that orally administered flavopiridol can strongly inhibit intimal formation to a level comparable to other clinically relevant agents<n>'<35>'<36> The oral activity of flavopiridol makes it unique among agents that have been shown to be active in animal models of vascular r damage. Its selectivity, potency and ease of administration make flavopiridol an excellent candidate for investigating the therapeutic benefits of cell cycle inhibition in vivo in human vascular lesions.

It was chosen to administer flavopiridol orally at a concentration of 5 mg/kg, half the dose that inhibits tumor growth in a nude mouse xenograft model 21. It is noteworthy that flavopiridol concentrations of 75 nmol/1 result in almost complete inhibition of SMC proliferation in the present studies, while mean serum concentrations of 425 nmol/1 were obtained at doses below the toxic limit in phase 1 human studies on refractory carcinoma<17>. Present results suggest that much lower doses of cell cycle inhibitors than those used in neoplasias can be effective in the area of vascular diseases, such as restenoses, with the simultaneous beneficial effect of increased tolerance.

While flavopiridol has been shown to induce growth arrest without affecting the viability of HASMC in culture, and has been shown to reduce neointimal formation after flavopiridol treatment in vivo, one cannot be sure that cell cycle arrest is the only factor that reduces neointimal formation in carotid lesions. Flavopiridol can induce growth arrest with or without inducing apoptosis, depending on the cell type observed<29>. Interestingly, flavopiridol inhibits apoptosis in terminally differentiated PC12 cells, while it induces apoptosis in undifferentiated PC12 cells that are proliferating<28>. Although the present in vitro experiments were performed under conditions that mimic the phenotype of SMC before injury, it is possible that SMC may respond differently to flavopiridol after injury and may even undergo apoptosis. While the role of apoptosis in vascular lesions is unclear, expression of the Fas ligand in SMC induces apoptosis and blocks neointimal formation in rats after balloon injury<37>, suggesting that if flavopiridol does indeed induce apoptosis in SMC in vivo, as it does in proliferating PC12- cells, this may be a favorable phenomenon within the framework of neointimal formation. Other mechanisms may also contribute to the effect of flavopiridol on lesion formation. For example, antisense oligodeoxynucleotide-mediated cell cycle inhibition improves endothelial function in rabbit vein grafts<38>. Further studies will be necessary to study the mechanisms of flavopiridol's effects, other than growth arrest, on SMC in vivo.

Given the demonstrated role of SMC proliferation in lesion formation after rat carotid injury<2>'<3>, it is interesting to note that, despite the clear effect of flavopiridol in vitro, its ability to inhibit neointimal formation, although the was significant; more moderately under conditions according to the present in vivo experiments. Several different explanations have been considered for this observation. It is unlikely that accelerated SMC proliferation occurs after cessation of flavopiridol, since differences in proliferative index persist as long as 14 days after injury (Figures 7 and 9). It is more likely that either other components of lesion formation, such as SMC migration and extracellular matrix production, still contribute to lesion formation, even in the absence of significant SMC proliferation, or that once-daily delivery of flavopiridol is insufficient to completely arrest proliferation in this model. Recent data suggest that the biological half-life of flavopiridol is as short as 2.5 hours, suggesting that the latter hypothesis may be correct; more studies may make it possible to identify an even more effective dose regimen<39>.

Present results suggest that flavopiridol can inhibit SMC proliferation and therefore neointimal formation in a well-accepted model of vascular disease in small animals. It must be pointed out that the relevance of inhibition of SMC proliferation is controversial in human vascular lesions, and may differ depending on the characteristics of the lesion and the time at which proliferation observations are made. The proliferative index of SMC in human atherectomy specimens is remarkably low<40>, although these specimens do not necessarily reflect proliferative changes at earlier, more critical, stages of lesion development. In addition, arterial remodeling independent of neointimal growth may account for a significant proportion of the luminal obstruction after angioplasty in humans<41>.1 In contrast, the mitotic activity index in SMC is far higher (25% PCNA-positive) in atherectomy specimens from human lesions with "in -stent" restenoses, which is consistent with the established role of SMC hyperplasia but not remodeling in this process<4>. As the placement of stents and the clinical problems of restenosis with stents increase, there will be a need for an effective method to arrest SMC hyperplasia and neointimal formation. Since flavopiridol is a potent, orally available drug with specific Cdk inhibitory activity, and since safe doses of flavopiridol are known for humans, flavopiridol can be considered as a pharmaceutical candidate for the prevention of stent restenosis in humans.

Methods and Results: Using cultured human aortic muscle cells, it was found that flavopiridol at concentrations as low as 75 nmol/1 resulted in almost complete inhibition of basic fibroblast growth factor and thrombin-induced proliferation. At this dose, flavopiridol inhibited cyclin-dependent kinase activity, as measured by histone Hi phosphorylation, but had no effect on MAP kinase activity. Induction of the cell cycle-related protein cyclin Di, proliferating cell nuclear antigen and phosphorylated retinablastoma protein was also blocked by flavopiridol. Flavopiridol had no effect on cell viability. To test whether flavopiridol had a similar activity in vivo when administered orally, neointimal formation in rat carotid arteries after balloon injury was investigated. Flavopiridol (5 mg/kg) administered by gavage reduced the neointimal area by 35% and 39%, 7 and 14 days after injury, respectively.

Conclusion: Flavopiridol inhibits SMC growth in vitro and in vivo. Its oral availability and selectivity for cyclin-dependent kinases make it a potential therapeutic tool in the treatment of SMC-rich vascular lesions.

REFERENCES

1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s.

Nature 1993; 362:801-9. 2. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. Lab. Invest. 1983; 49:327-333. 3. Clowes A, Schwartz S. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ. Res. 1985; 56:139-145. 4. Kearney M, Pieczek A, Haley L, Losordo DW, Andres V, Schainfeld R, Rosenfield K, Isner JM. Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 1997; 95: 1998 -2002. 5. Mintz GS, Hoffmann R, Mehran R, Pichard AD, Kent KM, Satler LF, Popma JJ, Leon MB. In-stent restenosis: the Washington Hospital Center experience. Am. J.

Cardiol. 1998; 81:7E-13E.

6. Califf RM. Restenosis: the cost to society. Am. Heart J. 1995; 130:680-684.

7. Sherr CJ. Mammalian Gi cyclins. Cell 1993; 73:1059-1065.

8. Hunter T. Braking the cycle. Cell 1993; 75:839-841.

9. Chen D, Krasinski D, Chen D, Sylvester A, Chen J, Nisen PD, Andres V. Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27<Kn>1>, an inhibitor of neointima

formation in the rat carotid artery. J. Clin. Invest. 1997; 99:2334-2341.

10. Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated overexpression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery

model of balloon angioplasty. J. Clin. Invest. 1995; 96:2260-2268.

11. Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel EG, Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular proliferative disorders with a constitutive

active form of the retinoblastoma gene product. Science 1995; 267:518-522.

12. Morishita R, Gibbons GH, Horiuchi M, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Pro. Nati.

Acad. Pollock. United States 1995; 92:5855-5859. 13. Losiewitz MD, Carlson BA, Kaur G, Sausville EA, Worland PJ. Potent inhibition of Cdc2 kinase activity by the flavanoid, L86-8275. Biochem. Biophys. Res. Commun.

1994; 201:589-595.

14. Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces Gi arrest with inhibition of cyclin-dependent kinase (CDK) 2 and cdk4 in

human breast carcinoma cells. Canc. Res.

15. de Azevedo WF, Mueller-Dieckmann H-J, Schuze-Gahmen U, Worland PJ, Sausville E, Kim S-H. Structural basis for specificity and potency of a flavanoid inhibitor of human Cdk2, a cell cycle kinase. Pro. Nati. Acad. Pollock. United States 1996;

93:2735-2740.

16. Kaur G, Stetler-Stevenson M, Sebers S, Worland P, Sedlacek H, Myers C, Czech J, Naik R, Sausville E. Growth inhibition with reversible cell cycle arrest of carcinoma

cells by flavone L86-8275. J. Nati. Canc. Institute 1992; 84:1736-1740.

17. Senderowicz AM, Headlee D, Stinson S, Lush RM, Figg WD, Pluda J, Sausville Ea. A Phase I trial of flavopiridol (FLA), a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasm. Pro. Am. Soc. Clin. Oncol. 1997; 16:226a.

Abstract.

18. Ruef J, Hu ZY, Yin L-Y, Wu Y, Hanson sr, Kelly AB, Harker LA, Rao Gn, Runge MS, Patterson C. Induction of vascular endothelial growth factor in ballooninjured

baboon arteries. Circ. Res. 1997; 81:24-33.

19. Ruef J, Rao Gn, Li F, Bode C, Patterson C, Bhatnagar A, Runge MS. Induction of rat aortic smooth muscle cell growth by the lipid peroxidation product 4-hydroxy-2-noneal. Circulation 1998; 97:1071-1078. 20. Swank Ra, T'ng Jp, Guo XW, Valdez J, Bradbury EM, Gurley LR. Four distinct cyclin-dependent kinases phosphorylate histone Hi at all of its growth related

phosphorylation sites. Biochemistry 1997; 36:13761-13768.

21. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimulating factor 1 regulates novel cyclins during the Gi phase of the cell cycle. Cell 1991; 65:701-713. 22. Bravo R. Synthesis of the nuclear protein (PCNA) and its relationships with DNA replication. Exp. Cell Res. 1986; 163:287-293. 23. Ewen ME, Sluss HK, Sherr CJ, Matsushime H, Kato JY, Livingston DM. Functional interaction of the retinoblastoma protein with mammalian D-type cyclins. Cell 1993; 7:487-497. 24. Hunter T. Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signalling. Cell 1995; 80:225-236. 25. Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J-H, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBOY. 1991; 10:885-892. 26. Konig A, Schwartz GK, Mohammad RM, Al-katib A, Gabrilove JL. The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia lines. Blood 1997; 90:4307-4312. 27. Drees M, Dengler WA, Roth T, Labonte H, Mayo J, Malspeis L, Grever M, Sausville EA, Fieberg HH. Flavopiridol (L86-8275): Selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. Clin. Cancer Res. 1997;

3:273-279.

28. Park DS, Rarinelli SE, Greene LA. Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated PC 12 cells and sympathetic

neurons. J. Biol. Chem. 1996; 271:8161-8169.

29. Parker BW, Kaur G, Nieves-Niera W, Taimi M, Kohlhagen G, Shimizu T, Losiewicz MD, Pommier Y, Sausville EA, Senderowicz AM. Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 1998;

91:458-465.

30. Geng YJ, Wu Q, Muszynski M, Hansson GK, Libby P. Apoptosis of vascular smooth muscle cells induced by in vitro stimulation with interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta. Arteriosclerosis. Thromb. Biol. 1996; 16:19-27. 31. Schwartz SM, deBlois D, 0'Brien ERM. The intima: soil for atherosclerosis and restenosis. Circ. Res. 1995; 77:445-465. 32. Wei GL, Krasinkski K, Kearney M, Isner JM, Walsh K, Andres V. Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty.

Circ. Res. 1997; 80:418-426. 33. de Young MB, Dichek DA. Gene therapy for restenosis. Circ. Res. 1997; 82:306-313. 34. Brooks EE, Gray NS, Joly A, Kerwar SS, Lum R, Mackman RL, Norman TC, Rosete J, Rowe M, Schow SR, Schultz TG, Wang X, Wick MM, Shiffman D. CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal formation. J.

Biol. Chem. 1997; 272:299207-29911.

35. Sirois MG, Simons M, Edelman ER. Antisense oligonucleotide inhibition of PDGFR-b receptor subunit expression directs suppression of intimal thickening. Circulation

1997; 95:669-676. 36. Rade JJ, Schulick AH, Virmani R, Dichek DA. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury.

Nature Med. 1996;2:293-298.

37. Sata M, Perlman H, Muruve DA, Silver M, Ikebe M, Libermann TA, Oettgen P, Walsh K. Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response. Pro. Nati. Acad. Pollock. USA

1998; 95:1213-1217.

38. Mann MJ, Gibbons GH, Tsao PS, von der Leyen HE, Cooke JP, Buitrago R, Kernoff R, Dzau VJ. Cell cycle inhibition preserves endothelial function in

genetically engineered rabbit vein grafts. J. Clin. Invest. 1997; 99:1295-1301.

39. Arguello F, Alexander M, Sterry JA, Tudor G, Smith EM, Kalavar NT, Greene JF, Koss W, Morgan CD, Stinson SF, Siforf TJ, Alvord WG, Klabansky RL, Sausville EA: Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity in vivo against humans

leukemia and lymphoma xenografts. Blood 1998; 91:2482-2490.

40. 0'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N, Gordon D, Benditt EP, Hinohara T, Simpson JB, Schwartz SM. Proliferation in primary and restenotic coronary atherectomy tissue: implications for anti-proliferative therapy. Circ. Res.

1993;73:223-231. 41. Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler LR, Wong SC, Hong MD, Kovach JA, Leon MB. Arterial remodeling after coronary angioplasty: a serial intravascular ultrasound study. Circulation 1996; 94:35-43.

Claims (6)

1. Use of a compound of formula Ia where R 1 is hydrogen, C 1 -C 3 alkyl, naphthyl, phenyl; phenyl mono- or poly-substituted with halogen, d-C4-alkyl, d-C4-alkoxy, hydroxyl, carboxyl, COO-Ci-C6- alkyl, CONH2, CONH-C1-C6- alkyl, CON(Ci-C6-alkyl) 2, nitro, trifluoromethyl, amino, C 1 -C 4 -alkylamino, di-C 1 -C 4 -alkylamino, or phenyl; pyridyl or thienyl; R 2 is hydrogen or C 1 -C 3 alkyl; R5 is C1-C3-alkyl, C3-C5-cycloalkyl, or C3-C5-cycloalkyl-C1-C4-alkyl, or a pharmaceutically acceptable acid addition salt thereof, for the preparation of a pharmaceutical preparation for inhibiting smooth muscle proliferation, where the dose of the compound according to formula Ia is less than 70%, preferably less than 60%, in particular less than 50% of the dosage that is necessary to control tumor growth. 2. Use according to claim 1, where the substituents in formula Ia have the following meaning: Ri is phenyl, thienyl, pyridyl, chlorophenyl, dichlorophenyl, methylphenyl, aminophenyl, bromophenyl, hydroxyphenyl or naphthyl; R2 is hydrogen and R5 is methyl. 3. Use according to claim 1, wherein the compound is (-)-cis, -5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl]-4H-benzopyran -4-one (Flavopiridol). 4. Use according to one or more of claims 1-3, where the pharmaceutical preparation is used for the treatment of vascular lesions rich in smooth muscle cells. 5. Use according to one or more of claims 1-3, where the pharmaceutical preparation is used for the treatment of lesions after balloon damage. 6. Use according to one or more of claims 1 - 4, where the pharmaceutical preparation is used to treat patients after stent implantation.